Process of making cationic lipid-nucleic acid complexes

ABSTRACT

The invention is related to a method for preparing an homogenous suspension of stable lipid-nucleic acid complexes or particles, comprising: a) combining one or more cationic lipids, one or more colipids, and one or more stabilizing additives to form a lipid suspension, b) combining the lipid suspension with a nucleic acid to form a complex or a particle, and optionally c) subjecting the complex or particle to a sizing procedure. It also concerns an homogenous suspension produced notably by the above method.

INTRODUCTION

The present invention is directed to stable complexes of cationic lipidsand nucleic acid that can be used to deliver nucleic acid to a cell forthe purpose of providing a therapeutic molecule to the cells of anindividual in need of such treatment, and to methods for the preparationof stable, cationic lipid-nucleic acid complexes.

BACKGROUND OF THE INVENTION

Successful gene therapy depends on the efficient delivery to andexpression of genetic information within the cells of a living organism.Most delivery mechanisms used so far involve viral vectors. Viruses havedeveloped diverse and highly sophisticated mechanisms to achieve thisgoal including crossing of the cellular membrane, escape from lysosomaldegradation, delivery of their genome to the nucleus and, consequently,have been used in many gene delivery applications.

Non-viral vectors, which are based on receptor-mediated mechanisms(Perales et al., Eur. J. Biochem. 226:255-266, 1994; Wagner et al.,Advanced Drug Delivery Reviews 14:113-135, 1994) or on lipid-mediatedtransfection (Eelgner et al., Proc. Natl. Acad. Sci. U.S.A. 84:7413-74171987; Behr et al., Proc. Natl. Acad. Sci. U.S.A. 86:6982-6986, 1989; Gaoet al., Biochem. Biophys. Res. Communic. 179:280-285, 1991; Behr,Bioconjugate Chemistry 5:382-389, 1994; Fahrhood et al., Annals New YorkAcademy of Sciences, 716:23-35, 1994; Ledley, Human Gene Therapy6:1129-1144, 1995) promise to have advantages with respect tolarge—scale production, reduced risks related to viral vectors,targeting of transfectable cells, lower immunogenicity and the capacityto deliver larger fragments of DNA.

The development of non-viral vectors for the delivery of nucleic acidsinto cells necessitates molecules that can associate with nucleic acidsto allow these large, hydrophilic polyanions to cross the cell membrane,which is a hydrophobic, negatively charged barrier of a phospholipidbilayer, help them to escape from lysosomal degradation and facilitatetheir entry into the nucleus. Expression of the gene of interest alsodepends on the accessibility of the delivered nucleic acid to thecellular transcription machinery, most likely necessitating dissociationof the complexes.

Although having been described as early as 1965 (Bangham et al., J. Mol.Biol. 13:238-252, 1965), liposomes, which can encapsulate molecules ofinterest for delivery to -he cell, did not reach the marketplace asinjectable therapeutics in humans until the 1990s (Gregoriadis, Trendsin Biotechnol. 13:527-537, 1995). This delay is attributable todifficulties in obtaining reproducible formulations with acceptablestabilities. A major breakthrough was the development of “stericallystabilized (stealth) liposomes” which contained a certain percentage ofpolyethyleneglycol (PEG)-modified phospholipids (PEG-PLs) (Gregoriadis,Trends in Biotechnol. 13:527-537, 1995; Lasic, Angew. Chem. Int. Ed.Engl. 33:1685-1698, 1994). These PEG-PL containing liposomes proved tobe more successful in evading detection and elimination by thereticulo-endothelial system, which resulted in greatly enhancedcirculation half-lives. While (PEG)-modified liposomes have been mostwidely used, other hydrophilic polymers such as poly(vinyl pyrrolidone)which augment the stability of liposomes when grafted on their surfacehave been described (Torchilin, V. P. et al., Biochim. Biophys. Acta1195:181-184, 1994).

Progress in lipid-mediated nucleic acid transfer into cells was advancedwith the introduction of cationic lipids as vehicles for the transfer ofnucleic acids into cells (Felgner et al., Proc. Natl. Acad. Sci.84:7413-7417, 1987; Behr et al., Proc. Natl. Acad. Sci. 86:6982-6986,1989; Gao et al., Biochem. Biophys. Res. Communic. 179:280-285, 1991;Behr, Bioconjugate Chemistry 5:382-389, 1994; Fahrhood et al., AnnalsNew York Academy of Sciences 716:23-35, 1994; Ledley, Human Gene Therapy6:1129-1144, 1995). Because cationic lipids are positively charged, theyare able to complex with negatively charged nucleic acids. Unlikeliposomes, these complexes do not require an encapsulation step and areprepared by simple mixing of components. The complexes essentiallycomprise of lipid-coated nucleic acid, in which the positively chargedcoat of the complex neutralizes the negative charges of the nucleic acidand, also, can efficiently bind to the negatively charged cell surface,facilitating entry of nucleic acid into the cell (Farhood et al., AnnalsN.Y. Acad. Sci. 716:23-35, 1994).

The advantages of using cationic lipids to mediate transfection ofnucleic acids include the simplicity of preparation of the complexes,the ability of the lipid component to complex most of the nucleic acid,a wide range of cell types amenable to transfection, a high efficiencyof transfer, lack of immunogenicity of the complexes and availability ofthe cationic lipids through chemical synthesis (Farhood et al., AnnalsN.Y. Acad. Sci. 716:23-35, 1994).

Cationic lipid-mediated delivery of nucleic acid to a wide variety ofcell types has been demonstrated in vitro and in vivo. For example,nucleic acid encoding the cystic fibrosis transmembrane conductanceregulator (CFTR) complexed with cationic lipids has been delivered tomouse lungs by intratracheal installation (Yoshimura et al., NucleicAcids Res. 20:3233-3240, 1992) or by aerosol delivery (Stribling et al.,Proc. Natl. Acad. Sci. 89:11277-1281, 1992). The delivery of CFTR usingcationic lipids to a mouse model of cystic fibrosis (CF) producedcorrection of the ion channel defect (Hyde et al., Nature 362:250-255,1993). Human clinical studies with cationic lipid-mediated delivery ofthe CFTR gene to CF patients demonstrated expression of the gene innasal epithelium and no adverse clinical effects (Caplen et al., NatureMedicine 1:39-46, 1995).

Systemic gene expression of a reporter gene following a singleintravenous injection of a cationic lipid-DNA complex has been shown inmice (Zhu et al., Science 261: 209-211, 1993). Moreover, safety studiesin rodents and non-human primates of systemically administered cationiclipid-nucleic acid complexes have shown no significant toxicityassociated with administering such complexes (Parker et al., Human GeneTherapy 6:575-590, 1995).

Cationic lipid-mediated transfection of mRNA in tissue culture wasdemonstrated to lead to translation of the transcript (Malone et al.,Proc. Natl. Acad. Sci. 86:6077-6081, 1989). Delivery of antisenseoligonucleotides to human endothelial cells using cationic lipidsprovided increased cellular uptake of the oligonucleotides and increasedactivity thereof in the cells (Bennett et al., Mol. Pharm. 41:1023-1033,1992). The use of cationic lipids complexed to retroviral particles hasallowed viral infection of cells which lacked the appropriate virusreceptor (Innes et al., J. Virol. 64:957-962, 1990) or enhancedretroviral transduction using complexes known as virosomes (Hodgson etal., Nature Biotechnol. 14:339-342, 1996).

Immunotherapy for cancer using cationic lipid-nucleic acid complexescontaining major histocompatibility (MHC) genes directly injected intomice tumors elicited immune responses that resulted in tumor regression(Plautz et al., Proc. Natl. Acad. Sci. 90:4645-4649, 1993).

Human clinical studies are currently underway using cationiclipid-mediated delivery of DNA sequences encoding immunotherapeuticmolecules in human melanoma, colorectal and renal cancer patients (Nabelet al., Proc. Natl. Acad. Sci. 90:11307-11311, 1993; Crystal, Science270:404-410, 1995).

The cationic lipid formulations to date have often incorporated thephospholipid dioleoylphosphatidylethanolamine (DOPE). This phospholipidis thought to disrupt the endosomal membrane by destabilizing itsbilayer structure, allowing the lipid-nucleic acid complex to escapeendosomal degradation and to enter into the cytoplasm (Farhood et al.,Biochem. Biophys. Acta 1235:289-295, 1995).

However, a significant obstacle in the widespread use of cationic lipidcomplexes for nucleic acid delivery to cells is the tendency of thecomplexes to form large aggregates in solution.

SUMMARY OF THE INVENTION

The present invention is directed to stable complexes or particles ofcationic lipids and nucleic acid that can be used to deliver nucleicacid to a cell for the purpose of providing a therapeutic molecule tothe cells of an individual in need of such treatment. The invention isalso directed to stable complexes or particles of cationic lipids andnucleic acid which contain a stabilizing additive. The invention isfurther directed to methods for the preparation of homogenoussuspensions of stable cationic lipid-nucleic acid complexes or particlesby combining one or more cationic lipids, one or more colipids, one ormore stabilizing additives and a nucleic acid or other ligand. Theinvention also includes a method for preparing an homogenous suspensionof stable cationic lipid-nucleic acid complexes or particles usingoptionnally sizing procedures such as extrusion, which can also be usedas the final sterilizing step in the production process of lipid-nucleicacid complexes for administration to patients for therapeutic purposes.

The invention is further directed to an homogenous suspension of stablelipid-nucleic acid complexes or particles produced by the above methods.

The invention may be understood with reference to the drawings of whichthe following is a brief description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic diagram of plasmid pCMVluc.

FIG. 2 shows recovery of plasmid DNA after extrusion of DNA vectorcomplexes containing spermidine-Chol/DOPE complexed to 200 μg/ml DNAthrough a 200 nm pore size. Percent yield is shown as a function of themolar percentage of distearoylphosphatidyl-ethanolamine-polyethyleneglycol (DSPE-PEG) and the charge ratio (+/−) of the complex.

FIG. 3 shows the integrity of pCMVluc plasmid DNA after extrusion oflipid-DNA complexes at 200 nm pore diameter determined by agarose gelelectrophoresis (lane 1: pCMVluc; lane 2: pCMVluc before extrusion ofcomplexes containing 200 μg/ml plasmid DNA complexed toSpermidine-Chol/DOPE (1:1 by weight) and 2 mol % DSPE-PEG2000 at acharge ratio +/−=5; lane 3: pCMVluc plasmid DNA in the same preparationafter extrusion at 0.2 μm; lane 4: the same preparation as shown in lane3 followed by extraction of the lipids to isolate the completed DNA).The spot at the bottom of the gel corresponds to carrier tRNA which isused to coprecipitate the plasmid DNA.

FIGS. 4(A-D) shows the effect of the molar percentage of DSPE-PEG2000 onthe stability of lipid-DNA complexes at 4° C. for different cationiclipids at 20 μg/ml DNA concentration as a function of time, as measuredby photon correlation spectroscopy after or without extrusion step.Black columns show the size of different DNA-cationic lipids complexescontaining 10% of DSPE-PEG2000 prepared without extrusion step; whitecolumns show the size of different DNA-cationic lipids complexescontaining 10% of DSPE-PEG2000 after extrusion; and grey columns showthe size of different DNA-cationic lipids, complexes containing 0% ofDSPE-PEG2000 and prepared without extrusion step. A: DC-Chol/DOPE; B:Spermidine-Chol/DOPE; C: Spermine-Chol/DOPE; D: DOGS/DOPE.

FIG. 5 shows the effect of different concentrations of DSPE-PEG2000 andof different +/− charge ratios on the stability of lipid-DNA complexescontaining spermidine-Chol/DOPE at a 200 μg/ml DNA concentration afterextrusion as a function of time. Black columns show +/−:5 charge ratioseffect; white columns show +/−:2,5 charge ratios effect; and greycolumns show +/−:1 charge ratios effect. A: 10% DSPE-PEG2000, B: 5%DSPE-PEG2000, C: 2% DSPE-PEG2000.

FIG. 6 shows the effect of different concentrations of DSPE-PEG2000 andof different +/− charge ratios on the stability of lipid-DNA complexescontaining spermidine-Chol/DOPE at a 200 μg/ml DNA concentration beforeextrusion as a function of time. Black columns show +/−:5 charge ratioseffect; white columns show +/−:2,5 charge ratios effect; and greycolumns show +/−:1 charge ratios effect. A: 10% DSPE-PEG2000, B: 5%DSPE-PEG2000, C: 2% DSPE-PEG2000.

FIG. 7 shows the effect of DSPE-PEG2000 on the in vitro transfectionactivity of lipid-DNA complexes in A549 cells as a function of plasmidDNA concentration. A) Expression level in the presence of 10 mol %DSPE-PEG2000 with respect to a buffer blank and B) in comparison to apreparation without DSPE-PEG2000.

FIG. 8 shows luciferase activity (RLU/mg protein) after intratrachealinjection of different cationic lipid-DNA complexes into mice as afunction of the day post-injection, compared to the injection of freeDNA only or an recombinant adenovirus control.

FIG. 9 shows luciferase activity (RLU/mg protein) after intratrachealinjection of spermidineChol/DOPE-pCMVluc complexes+10% DSPE-PEG2000 +/−extrusion into mice. Tissue sites of assay for each numbered mouse areas follows: (T:trachea; PG: left lung; PD: right lung).

FIG. 10 shows the influence of charge ratios and DSPE-PEG2000 (PEG-PL)on luciferase activity (RLU/mg protein) after intravenous injection ofcationic lipid-DNA complexes containing DC-Chol/DOPE into mice, comparedto the injection of free DNA. Tissue sites of the assays are shown.

FIG. 11 is a schematic representation of the synthesis of cationicglycerolipid pcTG56.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to stable complexes or particles ofcationic lipids and nucleic acid that can be used to deliver nucleicacid to a cell for the purpose of providing a therapeutic molecule tothe cells of an individual in need of such treatment. The invention isalso directed to stable complexes or particles of cationic lipids andnucleic acid which contain a stabilizing additive. The invention isfurther directed to methods for the preparation of homogenoussuspensions of stable cationic lipid-nucleic acid complexes by combiningone or more or mixtures of cationic lipids, one or more colipids, one ormore stabilizing additives and a nucleic acid or other ligand. Theinvention includes a method for preparing a homogenous suspension ofstable nucleic acid complexes or particles , comprising combining one ormore cationic lipids, one or more colipids, and one or more stabilizingadditives to form a lipid suspension, combining the lipid suspensionwith a nucleic acid to form a complex or a particle, and optionnalysubjecting the complex or particle to a sizing procedure to formcomplexes or particles of homogenous size. The invention also includes amethod for preparing an homogenous suspension of stable cationiclipid-nucleic acid complexes or particles using sizing procedures suchas extrusion, which can also be used as the final sterilizing step inthe production process of lipid-nucleic acid complexes foradministration to patients for therapeutic purposes.

The complexes or particles of the invention include one or more cationiclipids, one or more colipids, one or more stabilizing additives, and anucleic acid or other ligand.

The present invention is also directed to an homogenous suspension ofstable lipid-nucleic acid complexes or particles, produced by:

a) combining one or more cationic lipids, one or more colipids, and oneor more stabilizing additives to form a lipid suspension,

b) combining the lipid suspension with a nucleic acid to form a complexor a particle, and optionally

c) subjecting the complex or particle to a sizing procedure.

The invention also includes the above homogenous suspension wherein thelipid suspension is further subjected to a sizing procedure such that asuspension of complexes or particles of homogenous size is produced.

“Stable complexes or particles” means that independently of their sizesaid complexes or particles do not form aggregates.

“Homogenous suspension” is notably the result of a suspension containingnon-aggregated complexes or particles.

According to the present invention homogenously sized complexes orparticles may advantageously be obtained by an optionnal step subjectingsaid complexes or particles to a sizing procedure. Cationic lipids ormixtures of cationic lipids which may be used in the complexes of theinvention include Lipofectin™ (a mixture of DOTMA(N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride) andDOPE, 1:1 by weight), GIBCO-BRL; DDAB(dimethyl-dioctadecylammoniu-bromide); DMRIE(1,2-dimyristyl-oxypropyl-3-dimethyl-hydroxyethyl ammonium bromide);spermidine-cholesterol (spermidine-Chol); spermine-cholesterol(spermine-Chol); DC-chol (3β[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol); Transfectam™ (DOGS, dioctadecylamidoglycylspermine),Promega; DOSPER (1,3-di-oleoyloxy-2-(6-carboxy-spermyl)-propylamide),Boehringer Mannheim; DOTAP(N-[1-(2,3-dioleoyloxy)propyl]-N,N,N,-trimethyl-ammoniummethylsulfate),Boehringer Mannheim; Tfx™ 50 (a mixture ofN,N,N′,N′,-tetramethyl-N,N′,-bis(2-hydroxyethyl)-2,3-dileoyloxy-1,4-butamediammonium iodide and DOPE),Promega; Lipofectaminetm (DOSPA), Lipofectace™ (a mixture of DDAB andDOPE, 1:2.5 by weight), GIBCO-BRL.

Preferably, the cationic lipids of the present invention are selectedfrom among spermidine-cholesterol, spermine-cholesterol, DC-chol, andDOGS. Most preferably, the cationic lipid is any of the isomers ofspermidine-cholesterol.

Colipids are added to the complexes in order to facilitate entry of thenucleic acid into the cell or to conjugate additives that increasestability. Colipids of the invention include neutral, zwitterionic, andanionic lipids.

A preferred colipid which may be added to the complexes in order tofacilitate entry of the complexes into the cell isdioleoylphosphatidylethanolamine (DOPE).

In a preferred embodiment, the colipid DOPE is complexed to the cationiclipid in order to facilitate transport of the complex across the cellmembrane and prevent endosomal degradation.

The ratios of cationic lipid to colipid (on a weight to weight basis)can range from 1:0 to 1:10. In preferred embodiments, the ratio rangesfrom 1:0.5 to 1:4.

Other colipids may also be added to the complexes of the invention toallow the attachment of stabilizing additives to the complex. Thecolipid can be a moiety that allows the stabilizing additive to beincorporated into the complex of the invention. Derivatization of thelipid with an additive allows the moiety to anchor the stabilizingadditive to the cationic lipid complex. The colipid can be conjugated toadditives which prevent aggregation and precipitation of cationiclipid-nucleic acid complexes.

Colipids which may be used to incorporate such additives to the cationiclipid-DNA complexes of the invention include, but are not limited to,zwitterionic or other phospholipids. Preferably, the colipid used toconjugate a stabilizing additive is a moiety capable of beingincorporated into the complexes of the invention. More preferably, sucha colipid is inert and biocompatible.

In a preferred embodiment, the phospholipiddistearoylphosphatidylethanolamine (DSPE) is derivatized with astabilizing additive and is the moiety capable of being incorporatedinto the cationic lipid-nucleic acid complexes of the invention.

In another embodiment of the invention, cationic lipids may besynthesized to contain stabilizing additives such as polyethylene glycol(PEG), which product would bind to nucleic acid in a complex of theinvention through electrostatic interactions.

Stabilizing additives can also be added to the complexes of theinvention to maintain the integrity of the complexes, to maintaincomplex stability during sizing procedures, and to increase shelf life.The additives are preferably bound to a moiety capable of beingincorporated into or binding to the complex, for example, a colipid.Such additives generally are selected from among hydrophilic polymers,which include, but are not limited to, polyethylene glycol,polyvinylpyrrolidine, polymethyloxazoline, polyethyl-oxazoline,polyhydroxypropyl methacrylamide, polylactic acid, polyglycolic acid,and derivatized celluloses such as hydroxymethylcellulose orhydroxyethylcellulose (PCT Publication No. WO94/22429, published Oct.13, 1994). Other stabilizing additives useful in the present inventioninclude perfluorinated or partially fluorinated alkyl chains,fluorinated phospholipids, fatty acids and perfluoroalkylatedphospholipids and polyglucoronic acids (Oku et al., Critical Reviews inTherapeutic Drug Carrier Systems 11:231-270, 1994).

Preferably, the phospholipid DSPE is derivatized with polyethyleneglycol (PEG) to form the stabilizing additive DSPE-PEG. More preferably,the molecular weight of PEG that may be used ranges from 300 to 20,000Da. In a still more preferred embodiment, PEG2000 is used as thestabilizing additive.

The PEG-lipid conjugate can be prepared by several methods, includinguse of the linker cyanure chloride (U.S. Pat. No. 5,225,212,incorporated herein by reference). Other activating methods can be used,including those which use carbonyldiimidazole (C═O), succinic anhydride(—CO—CH₂—CH₂—CO—), or tosylate:

—(CH₂—CH₂—O—)_(n-1)CH₂—CH₂—NH—PE.

A proposed general structure of additives is as follows(PE=phosphatidylethanolamine):

Where the polymer is polyethylene glycol:

CH₃O—(CH₂—CH₂—O)n—X—NH—PE YO—(CH₂—CH₂—O)_(n)—X—NH—PE

Average molecular weight: 300-10000 Da,(n=5-250)

X: linker (CO; cyanure chloride, see U.S. Pat. No. 5,225,212)

Y: ligand (peptide, carbohydrate, protein, digionucleotide, vitamin,receptor antagonist or agonist, . . . )

In another embodiment of the invention, a ligand may be coupled to thestabilizer or PEG moiety of a complex of the invention using a free —OHgroup. Such ligands include, but are not limited to peptides,carbohydrates, proteins, nucleic acids, vitamins, receptor antagonistsand receptor agonists. Where the ligand is a digionucleotide, aPEG-digionucleotide conjugate may be prepared. Methods to activate the—OH group for coupling of the ligand include those which usecarbonyldiimidazole, cyanure chloride, succinic anhydride or tosylate.Preferably, the ligand contains a free NH2 or SH group available forcoupling.

An example is the following:

Ligand—NH—CO—O—PEG—O—CO—NH—DSPE.

The coupling of the ligand may be done at any of several stages ofcomplex formation, including at the molecular level between DSPE-PEG-OHand the ligand, at the level of the lipid suspension, or at the level ofthe lipid-DNA complex. Preferably, the coupling is performed at a laterstage of complex formation, with an increased probability that theligand will be on the surface of the complex and therefore accessible.

Stabilizing additives which allow for increased storage of the cationiclipid-nucleic acid complexes may be added to the complexes so as toachieve such long-term stability, but must be added so that theadditives do not interfere with the binding of the nucleic acid to thecationic lipid, especially at lower charge ratios. The molar ratio of astabilizer additive, such as a derivatized phospholipid, to the colipid(for example, DSPE-PEG2000/DOPE) may range from 0.01 to 1 (0.5 mol % to50 mol % with respect to the total lipid amount). In a preferredembodiment, the stabilizer additive may be added into the mixture ofcationic lipid and colipid at concentrations of from 1 to 20 mol % oftotal lipid. In a more preferred embodiment, the molar ratio ofstabilizer additive to colipid may range from 0.04 to 0.2.

The complexes of the present invention also include a desired nucleicacid component which is to be delivered to a cell in need of such amolecule. The nucleic acid which is complexed with the lipid suspensionor cationic lipid may be a DNA or RNA molecule, which may be single ordouble stranded. The nucleic acid may be, inter alia, a genomic DNA, acDNA, an mRNA, an antisense RNA, or a ribozyme. The nucleic acid mayalso be in the form of a plasmid or linear nucleic acid which containsan expressible sequence of nucleic acid that can generate a protein,ribozyme, antisense, or other molecule of interest upon delivery to acell. The nucleic acid can also be an oligo-nucleotide which is to bedelivered to the cell, e.g., for antisense or ribozyme functions. In apreferred embodiment using expressible nucleic acid, plasmid DNA isused. Concentrations of nucleic acid which may be added to the cationiclipids or lipid suspensions to form the complexes of the invention rangefrom 10 μg/ml to 1000 μg/ml. In a preferred embodiment of the invention,the concentration of nucleic acid ranges from 20 μg/ml to 500 μg/ml.

The cationic lipid-nucleic acid complexes of the invention may also becharacterized by their charge ratio (+/−), which is the ratio of thepositively charged cationic lipid component to the negatively chargednucleic acid component of the complex. In general, an excess positivecharge on the complex facilitates binding of the complex to thenegatively charged cell surface. The charge ratio of a complex of theinvention may be calculated by dividing the sum of the positive chargesby the negative charges on the complex. A determination of the positivecharge per mole can be made for a specific cationic lipid or colipidsuch that a given weight of lipid will represent a specific positivecharge. An analogous determination can be made with respect to thenucleic acid or colipid to yield the negative charge per mole so that agiven weight of nucleic acid or colipid will represent a specificnegative charge. By dividing all positive charges by all negativecharges, the net charge ratio (+/−) of the complex is determined.

Preferably, the range of the charge ratio of the complexes of theinvention are from 1 to 20 (+/−). More preferably, the range is from 2.5to 10 (+/−).

The invention is also directed to methods for homogenous sizing of thelipid suspensions (before addition of nucleic acid) and/or lipid-nucleicacid complexes using sizing methods which standardize the particle sizeof the lipid suspensions or lipid-nucleic acid complexes. Extrusionbefore the addition of nucleic acid would reduce nucleic acid binding tolipid aggregates. Methods which can be used to produce homogenouspreparations or suspensions of the complexes include, but are notlimited to, extrusion, sonication and microfluidization, size exclusionchromatography, field flow fractionation, electrophoresis andultracentrifugation.

In a preferred embodiment of the invention, a method comprisingextrusion of lipid suspensions or/and lipid-nucleic acid complexesthrough membranes of defined pore diameter is used to preparehomogenously sized particles or preparations of the complexes of theinvention without modification of the complexed nucleic acid. Anextruder may be used in which polycarbonate membranes of defined porediameter are stacked so that the suspension is forced through themembranes under pressure (for example, from Lipex Biomembranes, Inc.,Vancouver, Canada). Lipid suspensions or/and cationic lipid-nucleic acidcomplexes may be extruded through membranes having pores of 50 to 500 nmdiameter. Preferred membranes have a pore diameter of 200 nm. Extrusionof the complexes can also be used as the final sterilizing step in theproduction process of cationic lipid-nucleic acid complexes foradministration to patients for therapeutic purposes.

In a preferred embodiment of the invention, the particle size of acationic lipid-nucleic acid complex may range from 25 to 500 nm. Morepreferably, the particle size of a complex is 200 nm. Particle size maybe selected for optimal use in a particular application. For example,where a particular clinical application involves extravasation of thecationic lipid-nucleic acid complexes, the complex size may be about 80nm or lower.

Measurements of particle size can be made by a number of techniquesincluding, but not limited to, dynamic laser light scattering (photoncorrelation spectroscopy, PCS), as well as other techniques known tothose skilled in the art (see Washington, Particle Size Analysis inPharmaceutics and other Industries, Ellis Horwood, New York, 1992, pp.135-169).

After the complexes of the invention have been subjected to a sizingprocedure, e.g., extrusion, the yield percentage may be calculated toassess the recovery. This calculation is based on determining theconcentration of nucleic acid in the complexes before and after theprocedure. For example, where a suspension of complexes containing DNAis extruded through sizing membranes, the DNA concentration in thesuspension may be determined using standard techniques, e.g.,measurement of absorbance at 260 nm (A260), which detects nucleic acid.The presence of 10% DMSO facilitates the determination of DNAconcentration in the presence of lipid. The yield percentage afterextrusion may be calculated from the ratio of the DNA concentrationsdetermined before and after extrusion.

To determine the structural integrity of the nucleic acid in thecomplexes following a sizing procedure, the nucleic acid may beanalyzed, for example, by agarose gel electrophoresis after solventextraction of the lipids. The use of restriction mapping of the nucleicacid may provide additional detail regarding the state of a plasmid, forexample. Using such techniques, it is possible to determine whether thenucleic acid in a complex remains structurally intact and is notdegraded by shear forces during pressurized filtration or othermechanical stresses generated in sizing procedures.

It is also possible to assess the structural stability of the complexesin terms of how tightly the DNA is bound and covered by the lipid orlipid stabilizer component of the complex. This may be measured byassessing DNA migration in an agarose gel (no migration indicates thatthe DNA is covered with the lipid) or by incubation with DNAseI followedby lipid extraction and agarose gel electrophoresis to assess whetherthe DNA in the complex was exposed to the surface.

The complexes of the invention may be stored at 4° C. for optimalstability. Stability of the complexes over time may be determined byperiodic assessment of particle size, using methods previously describedfor such measurement.

The cationic lipid-nucleic acid complexes are also useful for transferof nucleic acid into cells for the purpose of monitoring behavior of thenucleic acid in the cell environment. For example, cationic lipidtransfection of a gene into cells of interest may be used to determineregulatory parameters that allow expression of the gene-enhancers,promoters, etc.- by linking these elements to the gene of interest andassaying for expression.

The cationic lipid-nucleic acid complexes can be used for delivery ofthe nucleic acid to the cells of an individual in need of treatment bysuch molecules. Routes of administration include, but are not limited todirect injection (e.g., intratracheal), aerosolization, intramuscular,intra-tumoral and intravenous routes for in vivo administration.Cationic-lipid-mediated transfection of nucleic acid into cells used inex vivo transplantation procedures can also be used to deliver nucleicacid to an individual in need of such molecules.

Therapeutic transgenes that can be used as the nucleic acid component ofthe complexes of the invention include, but are not limited to, CFTR forcystic fibrosis, α1-antitrypsin for emphysema, soluble CD4 for AIDS, ADAfor adenosine deaminase deficiency, dystrophin for muscular dystrophy,cytokine genes for cancer treatment, immunotherapeutic or tumorsuppressor genes for cancer treatment, and any other genes that arerecognized in the art as being useful for gene therapy. The nucleic acidcomponent may also include mRNA. Transgenic nucleic acids encodingmolecules such as ribozymes, antisense RNA, or oligonucleotides can alsobe contained in the nucleic acid component of a complex of theinvention. Alternatively, ribozymes, antisense nucleic acid oroligonucleotides may be directly incorporated into the complexes of theinvention. The nucleic acid constructs may also contain regulatoryelements that govern expression of the gene, such as enhancers andpromoters.

Where complexes comprising an expressible nucleic acid are delivered toa wide variety of cell types, e.g., by various administration routesincluding intravenous administration, leading to systemic uptake of thelipid-nucleic acid complexes, expression of the nucleic acid may belimited to specific tissues by the use of tissue-specific promoters.Temporal control of expression of nucleic acid also may be achieved withthe use of inducible promoters that are activated in response to anexogenous stimulus, e.g., an MMTV promoter activated by metallothionin,a TAR/RRE comprising promoter activated in the presence of the TAT/REVproteins of HIV, or a hormone responsive promoter.

Where the nucleic acid component of the complex comprises an expressiblegene, the biological function thereof may be assayed by standardtechniques, including detection of mRNA by Northern blot or SI analysis,and/or detection of protein using Western blotting, immunoprecipitationor functional protein assay. The latter is particularly useful where agene encodes a suitable marker protein, e.g., luciferase orP-galactosidase.

The cationic lipid-nucleic acid complexes may transfer the nucleic acidof interest to cells in vitro for the purpose of determining thefunction of such nucleic acid, or for the purpose of providing suchcells with a nucleic acid that provides a therapeutic benefit, or forthe purpose of determining the efficiency and specificity of thecomplexes for nucleic acid delivery. Such cells may include, but are notlimited to, established cell lines, e.g., A549, NIH3T3, HeLa, as well asprimary cells or other cells known to those skilled in the art.

The nucleic acid of interest may also be delivered in vivo to cells ofan animal model which can be used to determine the efficiency andspecificity of transfer to the tissues of a whole organism. Such animalsinclude mice (e.g., C57 Black/10 or Balb/c), rabbits, and primates aswell as others. Animal models of human disease states may be used totest the efficacy of certain molecules for therapeutic treatment, e.g.,transgenic mice engineered to express a mutant CFTR gene as a model ofcystic fibrosis.

The present invention also encompasses pharmaceutical compositionscontaining the complexes of the invention which can be administered in atherapeutically effective amount to deliver a desired nucleic acid to anindividual needing therapy. The pharmaceutical compositions can includepharmaceutically acceptable carriers, including any relevant solvents.As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredient, its use in the therapeutic compositions iscontemplated.

The practice of the invention employs, unless otherwise indicated,conventional techniques of recombinant DNA technology, proteinchemistry, microbiology and virology which are within the skill of theart. Such techniques are explained fully in the literature. See, e.g.,Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley& Sons, Inc., New York, 1995.

The invention is illustrated by reference to the following examples.

EXAMPLE 1

Preparation of Cationic Lipid-Nucleic Acid Complexes

Cationic lipids DC-Chol (3β[N-(N′,N′-dimethyl-aminoethane)-carbamoyl]cholesterol) (synthesized according to Gao, X. and Huang, L., Biochem.Biophys. Res. Communic. 179:280-285, 1991); spermidine-cholesterol(spermidine-Chol) (synthesized at Transgene, S. A.) (Caffey et al., J.Biol. Chem. 270:31391-31396, 1995; French Application No. 96 01347, bothincorporated herein by reference) spermine-cholesterol (spermine-Chol)(synthesized at Transgene, S. A.) (Caffey et al., J. Biol. Chem.270:31391-31396, 1995; French Application No. 96 01347) anddiocta-decylamido-glycyl-spermine (DOGS) (generous gift of Dr. Jean-PaulBehr (Behr et al., Proc. Natl. Acad. Sci. 86:6982-6986, 1989,incorporated herein by reference), and colipidsdioleoylphosphatidylethanolamine (DOPE) (Sigma, Ref. P5058, lot 75H8377)and distearoyl-phosphatidyl-ethanolamine-PEG2000 (DSPE-PEG2000, AvantiPolar Lipids, Alabaster, Ala., USA, Ref 880120, lot 18OPEG2PE-21) werecombined at the required ratios (ratios of cationic lipid:DOPE of 1:1;ratio of DSPE-PEG2000 to total lipid of 2, 5 and 10 molt) by mixing therespective solutions in chloroform/ethanol (8:2) and evaporating thesolvents under a stream of nitrogen to produce a dried lipid film.Residual solvents were evaporated under vacuum. The dried lipid film wasrehydrated at 4° C. overnight with slight agitation in 20 mM Hepes, pH7.8, 0.9% NaCl and suspended by sonication for 8 min in a bath sonifier(Bransonic 221).

The cationic lipid/colipid mixtures were extruded at 200 nm beforecomplexing them with DNA at different charge ratios +/−. Extrusion wasperformed using an extruder from Lipex Biomembranes, Inc. (Vancouver,Canada) equipped with a stack of 2 polycarbonate membranes having poresof 0.2 μm diameter (Nucleopore, Costar Corp. Cambridge, Mass., USA). Thesuspension was forced through the membranes under nitrogen gas pressureof approximately 10 bar at 50° C.

In a particular example, the following lipids were mixed and dried: 422μl of Spermidine-Chol (10 mg/ml), 422 μl DOPE (10 mg/ml) and 145 μlDSPE-PEG2000 (25 mg/ml) under a stream of nitrogen, followed byevaporation of residual solvent under vacuum. Reconstitution wasachieved by adding 4.32 ml 20 mM Hepes, pH 7.8+0.9% NaCl overnight at 4°C. on a shaker, followed by sonication in a bath sonifier (Bransonic221) for 8 min to give the following lipid concentrations:spermidine-Chol: 1.75 mM, DOPE: 1.31 mM, DSPE-PEG: 0.31 m-M. The lipidsuspension was extruded through a stack of 2 membranes of 200 nm porediameter. The suspension was forced through the membranes under nitrogengas pressure of approximately 10 bar at 50° C. 200 μl pCMVluc plasmidDNA was added (1.46 mg/ml in 10 mM Tris-HCl, pH 7.5, 1 mM EDTA) to analiquot of 1.26 ml of the lipid suspension to reach a final volume of1.46 ml. The concentrations of the components in the final preparationof the complex were: spermidine-Chol: 1.51 mM; DOPE: 1.13 mM; DSPE-PEG:0.27 mM; DNA: 200 μg/ml. Such a complex contains 10 mol % DSPE-PEG2000and has a charge ratio of 5 at a DNA concentration of 200 μg/mi.

The lipid-DNA complexes were again extended through a 200 nm porediameter polycarbonate membrane at 50° C. and approximately 10 barnitrogen pressure. See FIG. 4 for comparison of the stability ofextruded and non-extruded lipid-DNA complexes.

The plasmid containing the luciferase gene under the control of the CMVpromoter, pCMVluc is shown in FIG. 1.

The particle size of the cationic lipid-nucleic acid complexes that wereobtained was determined by photon correlation spectroscopy (also calleddynamic light scattering), a technique which is based or laser lightscattering. PCS measures the Brownian movement of particles in theilluminated volume of the laser beam and calculates a correlationfunction which links the fluctuations in scattered light to thediffusion coefficient of the particles. The particle size is thenderived from the diffusion coefficient using the Stokes-Einsteinrelationship: D=kT/3πηd (k: Boltzmann constant; T: absolute temperature;η: viscosity; d: particle diameter (see Clive Washington, Particle SizeAnalysis in Pharmaceutics and other Industries, Ellis Horwood publisher,New York, 1992, pp 135-169; contains also chapters on other methods).

PCS was performed on an aliquot before extrusion, while the remainderwas extruded through a 200 nm membrane at 50° C. Particle diameter wasagain determined by PCS after extrusion. The cationic lipid-nucleic acidcomplexes were stored at 4° C. for various time periods to determinetheir stability.

The concentration of DNA in the cationic lipid-nucleic acid preparationswas determined by clarifying the lipid suspension with 10% (v/v)dimethylsulfoxide (DMSO). Absorbance was measured at 260 nm using therelationship that defines 50 μg/ml DNA as equal to 1 absorbance unit ata path length of 1 cm. Measurements were performed before and afterextrusion for the different preparations and compared with theconcentration of non-complexed plasmid DNA. The yield percentage iscalculated by taking the ratio A260 (after extrusion)/A260 (beforeextrusion) (×130).

The physical integrity of DNA in the complexes was determined by agarosegel electrophoresis after solvent extraction of the lipids. To this end,1 ml of cationic lipid-nucleic acid suspension was mixed with 0.4 mlwater, 2 ml methanol and 1 ml chloroform and vortexed for 2 min. Aftercentrifugation for 5 min at 3,000 rpm the upper phase was transferred toa separate tube and dried under vacuum. The pellet was redissolved in250 pi of water to which 27 μl 3M sodium acetate and 5 μg transfer RNAwere added, followed by the addition of 700 μl absolute ethanol that hadbeen cooled to −20° C. After brief vortexing and 1 h at −20° C., the DNApellet was recovered by centrifugation at 15,000 rpm for 30 min at 4° C.The liquid was decanted and the remaining pellet was washed twice with200 μl of 70% aqueous ethanol (cooled to −20° C.) and recovered bycentrifugation after each wash. The pellet was then dried under vacuumand redissolved in 10 mM Tris-HCl, pH 7.5, 1 mM EDTA to an approximatefinal concentration of 0.5 μg/pl. Agarose gel electrophoresis wasperformed in submarine slab gels (14×10×0.8 cm) in 1% agarose (Sigma,Ref. A-6877, lot 123HO552) in Tris (4.86 g), sodium acetate×3 H20(0.68g) and EDTA (0.336 g, all weights are for 1 liter final volume, adjustedto pH 7.8 with acetic acid) at 60 volts for 2 h. DNA bands were stainedwith ethidium bromide in the above buffer at 60 μg/l.

Particle size analysis was performed by laser light scattering (10 mWHe—Ne laser at 632.8 nm) combined with PCS (Coulter N4 Plus, Miami,Fla., USA) over the range of 3-10,000 nm. Scattered light was measuredat an angle of 90° of the sample diluted in 20 mM Hepes, pH 7.8, 0.9%NaCl to obtain between 5×104 and 106 counts/sec. The final volume was500 μl. The measurement was started after 3 min equilibration at 25° C.using the following parameters: automatic prescale; sample time:automatic; run time automatic (3 min at 900); SDP (algorithm todiscriminate between different populations) analysis from 3 to 10,000 nmusing 25 bins; refractive index: 1.33252; viscosity: 0.8904 centipoise.

After a first analysis performed under the above conditions, themeasurement conditions were refined by reducing the size of the windowand by increasing the number of bins. The system was calibrated withlatex beads of defined average particle diameter (Coulter size controls:CEI NO. 6602336; 90, 170, 300 and 500 nm). Adjustment of the delay timewas done automatically and was regularly verified to be in the correctrange by manual measurements on the calibrated beads.

Results

Incorporation of PEG-phospholipids (PEG-PL) such asdistearoylphosphatidylethanolamine coupled to PEG2000 (DSPE-PEG) atmolar ratios of 10% significantly improved DNA recovery after extrusionwhile maintaining its integrity by preventing aggregation (FIGS. 2 and3). This beneficial effect was already apparent upon visual inspectionof the obtained complexes which were homogenous dispersions in the caseof added PEG-PLs but became rapidly flocculent if the stabilizeradditive was omitted. Initial attempts to extrude lipid-nucleic acidcomplexes which contained no stabilizing additives through polycarbonatemembranes with a pore diameter of 200 nm had shown that most of the DNAwas lost on the filter membrane most likely because of the presence ofaggregates.

Stability studies using various cationic lipids complexed to 20 μg/mlplasmid DNA at 4° C. showed that the average particle diameter of thecomplexes as determined by PCS remained stable for at least 2 months(FIGS. 4A-D; A: DC-Chol/DOPE; B: Spermidine-Chol/DOPE; C:Spermine-Chol/DOPE; D: DOGS/DOPE). Initial particle size is shown at day0, and the particle size was measured over a 63 day period, as afunction of the mol % of DSPE-PEG 2000 and whether extrusion of theDNA-lipid complexes was performed (“ex”) The data shows that theaddition of 10% DSPE-PEG maintained the particles at uniform size,preventing aggregation, in contrast to the preparations without DSPE-PEG(0%).

The stabilizing effect of DSPE-PEG was also observed for lipid-DNAcomplexes containing spermidine-Chol/DOPE complexed to higher plasmidDNA concentrations (200 μg/ml) that are necessary for in vivotransfections (FIG. 5). The results show that the stabilizing effect ofDSPE-PEG2000 is observed for different amounts of the additive (2, 5 and10 mol %) as well as for different +/− charge ratios. Minor fluctuationsin the measured size by PCS do not indicate instabilities of thepreparations, since they do not evolve with time. It is more likely thatsize estimations by PCS may have introduced theses fluctuations,possibly due to slight changes in the shape of the complexes.

The particle size of lipid-DNA complexes containing PEG-PLs was alsostabilized when omitting the final extrusion step (FIG. 6) Thisstabilizing effect was apparent at different +/− charge ratios anddifferent concentrations of PEG-PLs. In particular, lipid-DNA complexeswith high +/− charge ratios were efficiently stabilized, but all of thepreparations had mean particle diameters of less than 400 nm and, inmost cases, of less than 200 nm as compared with particle sizes of morethan 1000 nm in the absence of PEG-PLs (FIGS. 4A-D). The absence ofPEG-PL in the complexes resulted in visible flocculation andprecipitation after addition of plasmid DNA to the cationic lipids at afinal concentration of 200 μg/ml, which prevented meaningful PCSmeasurements.

Stability studies at 4° C. showed that the extruded complexes remainedstable at an average particle diameter of about 170 nm (PCS measurement)for at least 2 months. Furthermore, such complexes allow in vivotransfection via the intratracheal and intravenous route as shown inExample 3. The additives stabilize complexes between cationic lipids andnucleic acids, and allow extrusion of such complexes through membranesof defined pore size without modification of the complexed nucleic acid.Phospholipids derivatized with polyethylene glycol prove to be efficientin preventing aggregation and precipitation of lipid-DNA complexes.

EXAMPLE 2

Transfection of Cells Using Cationic Lipid-Nucleic Acid Complexes

Methods

From an initial 20 ml of cell culture containing 2×106 cells, 2×104cells per well (96 well culture plate) were plated in 200 μl DMEM mediumsupplemented with glutamine and 10% fetal calf serum on day 1. On day 2,two microtiter plates were prepared: one plate contained differentdilutions of plasmid DNA in 70 μl medium without serum per well(starting concentration: 0.16 mg/ml) and one plate contained the lipidsuspension in 60 μl per well (starting concentration: 0.187 mg/ml). Boththe DNA and the lipids were serial diluted (factor of 2) and therequired amount of DNA was transferred into the well containing therequired amount of lipids. The medium was removed from the cells byaspiration and the lipid-DNA complexes (100 μl) were transferred ontothe cells. After 4 h at 37° C., 5% CO2 and 50 μl of medium containing30% fetal calf serum were added. On day 3, an additional 100 μl mediumcontaining 10% fetal calf serum were added and on day 4 the cells wereinspected microscopically for viability. The transfection was stopped byremoving the medium and the cells were washed with 100 μlphosphate-buffered-saline (PBS). After addition of 50 μl lysis buffer(Promega, 5× diluted in water) cells were frozen to −80° C. for at least15 min. The amount of luciferase produced was determined in 20 μl of thelysis solution for 1 min using the Luciferase Assay System (Promega) in96 well microtiter plates (Berthold) in the kinetics mode of a BertholdLB 96 P luminometer.

Results

Incorporation of 10 mol % of PEG-PLs completely blocked the in vitrotransfection activity of the lipid-DNA complexes in cultured A549 cells(FIGS. 7A and B). This effect may be due to the fact that the PEG-PLsprevent efficient contact between the vector complexes and cells inculture. It was thus even more surprising to find that these vectorsallowed transfection in vivo by either the intratracheal (i.t.) orintravenous (i.v.) route of administration (see results below).

EXAMPLE 3

In Vivo Administration of Cationic Lipid-Nucleic Acid Complexes

Methods

Intratracheal injection into mice: 5 to 6 weeks old mice (C57 Black/10or Balb/c) were anesthetized using Ketamine and 125 μl vectorpreparations containing 25 μg of pCMVluc plasmid DNA complexed tocationic lipids at different charge ratios were injected into thetrachea. Complexes were not extruded and contained no DSPE-PEG2000.After 48 hours, the mice were sacrificed and the trachea, left and rightlungs were removed, frozen in liquid N2 and stored at −70° C.

Intravenous injection into mice: 5 to 6 weeks old mice (C57 Black/10 orBalb/c) were injected with 400 μl vector preparations into the tailvein. Preparations contained 75 μg pCMVluc DNA complexed to cationiclipids at different charge ratios. After seven days, organs (lung,heart, spleen, liver, skeletal muscle) were taken, frozen in liquid N2and stored at −70° C.

Preparation of Protein Extracts and Determination of LuciferaseActivity:

Luciferase extracts were prepared as described (Manthorpe, Human GeneTherapy 4:419-43, 1993) with the following modifications. Frozen tissuewas pulverized in pre-cooled mortars on dry ice. The powder wastransferred to 1.5 ml Eppendorf tubes and extracted in 500 pi ReporterLysis Buffer (Promega) using 3 cycles of freeze-thaw in liquid N2 and at37° C. Lysates were centrifuged at 14,000 rpm for 10 min in an Eppendorfcentrifuge at room temperature and supernatants were transferred to newtubes. The pellet was eventually used for DNA extraction. Extracts werefrozen in liquid N2 and stored at −70° C. Protein concentrations weredetermined with the Quantify Protein Assay System (Promega). Luciferaseactivity was measured in 10 μl aliquots of extracts using the LuciferaseAssay System (Promega) in 96 well microliter plates (Berthold) using thekinetics mode of a Berthold LB 96 P luminometer. Luciferase activitieswere measured on organ samples, i.e., trachea, left and right lung,which were taken from injected mice. After hybridization and lysis inreporter lysis buffer (Promega), 10 or 20 μl aliquots are mixed with 100μl of substrate (Luciferase Assay System, E1501, Promega). One minutereadings were taken on the luminometer according to the manufacturer'sprotocol. Luciferase activities were calculated either as RLU/mg proteinor as fg luciferase/mg protein with the aid of a luciferase standardcurve, that was established with purified enzyme (Promega) diluted in anegative tissue extract.

Results

Intratracheal administration: FIG. 8 summarizes an experiment withdifferent cationic lipids (DC-Chol, spermidinechol, spermine-chol)formulated with the colipid DOPE that were used to complex pCMVluc DNAat a charge ratio of 5:1. The ratio of cationic lipid:DOPE is 1:1 byweight. Lipid-DNA complexes in this experiment were prepared andirrimediately injected i.t. into the test animals to avoid the formationof large aggregates that could reduce the transfection efficiency of thecomplexes. Luciferase activities were measured as previously describedon organ samples, i.e., trachea, right and left lung taken from injectedmice 1, 2 and 3 days after i.t. injection, using a recombinantadenovirus containing the luciferase gene as a control.

Luciferase activity could be detected for DC-Chol/DOPE, orspermine-Chol/DOPE complexed DNA at 24 and 48 hrs. The same was true foradenovirus in 0.9% NaCl. No activity was found for free DNA and in thisexperiment for DNA complexed to spermidine-Chol/DOPE.

Extruded complexes with additives: The next set of experiments testedlipid-DNA formulations having a defined particle size and an increasedstability after formulation. For this purpose, complexes were preparedby extrusion which generates particles with an average size of about 200nm. The addition of DSPE-PEG2000 as a component of lipid-DNA complexesin these formulations was also tested.

Spermidine-Chol/DOPE was complexed with 25 pg pCMVluc at a charge ratioof 5:1 in the presence of 10 mol % DSPE-PEG2000. The ratio of cationiclipid:DOPE is 1:1 by weight. Samples were either directly administeredby intratracheal injection into 6 weeks old C57 Black/10 mice or sizefractionated by extrusion prior to injection. After 48 hours, animalswere sacrificed and luciferase activity was determined in trachea (T),left (PG) and right (PD) lungs of each animal. FIG. 9 shows luciferaseactivities after injection of spermidine-Chol/-DOPE-pCMvluc complexesinto mice. Mouse 11 was injected with 25 pg free pCMVluc DNA. Noluciferase activity could be detected after 48 h. Mice 13 and 14received complexes that were not extruded and mice 17, 18, 19 and 20received complexes after extrusion. Luciferase activities in the rangeof 1000 to 7000 RLU/mg protein were measured for tracheas and lungs ofall mice with the exception of mouse 18 that showed no activity intrachea.

Effect of charge ratio and stabilizing additive: The influence of thecharge ratio of spermidine-Chol/DOPE-pCMVluc complexes and theconcentration of DSPE-PEG2000 in these complexes was further studied.Spermidine-Chol/DOPE-pCMVluc complexes with charge ratios 5:1, 2.5:1 and1:1 were prepared. For each charge ratio DSP -PEG2000 was added to 10, 5or 2 mol %. 48 h after i.t. injection into C57 Black/10 mice, luciferaseactivities were determined in tracheas and lungs of these animals. Table1 summarizes the results of this experiment.

TABLE 1 Influence of Charge Ratios and DSPE-PEG2000 Concentration oni.t. activity of Spermidine-Chol/DOPE- pCMVluc complexes DSPE- Trachea*Lung* PEG2000 RLU/mg protein RLU/mg protein RATIO mol % +/− SD +/− SD5:1 10 4416 +/− 1472 1839 +/− 491 5 7200 +/− 1086 4173 +/− 34  2 0 8251+/− 635 2.5:1   10 12007 +/− 473  0 5 15263 +/− 911  0 2 7636 +/− 1192 01:1 10 77687 +/− 4956  0 5 378373 +/− 46162  0 2 116596 +/− 213   0*RLU/mg protein SD of at least three independent measurements of atleast two independent mice

Luciferase activity in lungs was measurable after injection of complexeswith a 5:1 charge ratio. A decrease of DSPE-PEG2000 from 10 to 2 moleresulted in an increase in luciferase activity within this experiment.At lower charge ratios, luciferase activity was no longer detected inthe lungs. In tracheas, luciferase activity increased with decreasingcharge ratios. The DSPE-PEG2000 concentration is probably alsoinfluencing this activity.

One explanation for this is that DSPE-PEG2000 may destabilize thecationic lipid-DNA complexes with respect to how tightly the DNA isbound to and covered by the cationic lipid. This effect would be moreimportant for lower charge ratios. Due to this effect, only complexeswith a high charge ratio are stable enough to reach and transfect lungcells. At lower charge ratios, the complexes become unstable and canefficiently transfect cells in the trachea next to the injection sitebut do not penetrate deep enough into the lung to transfect lungtissues.

Intravenous Administration:

To test the influence of different charge ratios and the presence ofDSPE-PEG2000 on formulations for i.v. delivery, 75 μg pCMVluc DNA werecomplexed at charge ratios 1:2, 1:4 and 1:6 with DC-Chol/DOPE in thepresence or absence of 10% DSPE-PEG2000 and i.v. injected in 400 μlvolumes. The ratio of cationic lipid:DOPE was 1:1 by weight. Thecomplexes were not extruded.

Luciferase activities were determined seven days later in lung, liver,heart, skeletal muscle and spleen. The results are shown in FIG. 10.Luciferase activity is indicated as RLU/mg protein +/− (standarddeviation) for groups of 4 independent mica per formulation. Noluciferase activity can be found in muscle and spleen. Low activitiesare found in heart and liver for charge ratios 1:4 (800 μg total lipid(400 μg DC-Chol):75 μg DNA) and 1:6 (1200:75). In lung relatively highvalues are detected starting at 1:2 (400:75) charge ratio +10%DSPE-PEG2000.

The ratio of lipids to DNA was changed in these experiments to evaluatethe influence of this parameter as well as the presence of DSPE-PEG2000on in vivo transfection. The rationale behind increasing the amount oflipids was that the DNA might be more stable in vivo and that a morepositive charge ratio might lead to a modified tropism of the complexes.It appears from this experiment that higher charge ratios lead to moreconsistent transfections. It is also noteworthy that the presence ofDSPE-PEG2000 prevented visible precipitations which formed in allpreparations that did not contain the additive.

EXAMPLE 4

Preparation of Cationic Lipid-Nucleic Acid Complexes Containing CationicGlycerolipids pcTG56

A. Synthesis of Cationic Glycerolipids pcTG56

PcTG56 has been prepared according to the following protocol (see alsoFIG. 15)

Cyano Acid 1

A solution of acrylonitrile (9.6 ml, 146 mmoles) in 1,4-dioxane (50 ml)was added dropwise to an ice-cold solution of glycine (10.0 g, 132mmoles) and of 1N sodium hydroxide (133 ml) in a 1/1 mixture of waterand of 1,4-dioxane (200 ml). The reaction was stirred at 0° C. for 1 hand at room temperature for an extra 4 h. A solution of ditertiobutyldicarbonate (35.0 g, 159 mmoles) in 1,4-dioxare (100 ml) was then addeddropwise and the reaction mixture was stirred for 2 h at roomtemperature. After extraction with ether (2×100 ml), the aqueous phasewas acidified (pH 2-3) with 1 N hydrochloric acid and extracted withethyl acetate (2×100 ml). The combined organic phases were dried withsodium sulfate and concentrated in vacuo. The cyano acid 1 (24.4 g; 81%yield) was obtained as a white solid which was used without furtherpurification. mp=87-89° C.

¹H NMR (200 MHz, DO): d 3.88 and 3.87 (2 s, 2H, —CH₂—CO₂H), 3.48 and3.45 (2 t, J=6.3 Hz, 2H, —CH₂—N(BOC)—), 2.58 and 2.56 (2 t, J=6.3,6.4Hz, 2H, —CH₂—CN), 1.30 and 1.24 (2 s, 9H, t—Bu—).

Amino Acid 2

A solution of cyano acid 1 (11.5 g, 50.4 mmoles) in ethanol (100 ml)containing sodium hydroxide (4.04 g, 100 mmoles) was hydrogenated in thepresence of Raney nickel (3.2 g) for 18 h at room temperature. Themixture was carefully filtered on celite and the catalyst washed withmethanol (2×30 ml). The filtrate was acidified (Ph 4-5) with 10% aqueoushydrochloric acid and concentrated in vacuo to give a white solid whichwas dissolved in chloroform (50 ml) to precipitate most of the sodiumchloride. After filtration, concentration in vacuo of the filtrate, andrecrystallisation in carbon tetrachloride, amino acid 2 (10.4 g; 89%)was obtained. mp=201-202° C.

¹H NMR (200 MHz, D₂O): d 3.53 (s, 2H, —CH₂—CO₂H), 3.17 (t, J=6.6 Hz, 2H,—CH₂—N(BOC)—), 2.83 (t, J=7.5 Hz, 2H, —CH₂—NH₂), 1.69 (quint., J=7 Hz,2H, —CH₂—), 1.26 and 1.21 (2 s, 9H, t—Bu—).

Cyano Acid 3

The same procedure as for 1 allowed the obtention of the cyano acid 3from 2.

¹H NMR (200 MHz, CDCl₃): d 4.00-3.85 (m, 2H, —CH₂—CO₂H), 3.55-3.43 (m,2H, —CH₂—CH₂—CN), 3.31 (t, J=7.2 Hz, 4H, —CH₂—N(BOC)—), 2.61 (m, 2H,—CH₂—CN), 1.78 (quint., J=7.2 Hz, 2H, —CH₂—), 1.47 and 1.44 (2 s, 18H,t—Bu—).

Amino Acid 4

The amino acid 4 (87% yield; purification by chromatography on a silicagel column, eluent methanol/dichloromethane 3/7, then 6/4) was obtainedfrom 3 by the same procedure as the amino acid 2. mp=189-190° C.

¹H NMR (200 MHz, D₂O): d 3.57 and 3.54 (2 s, 2H, —CH₂—CO₂H), 3.2-3.0 (m,6H, —CH₂—N(BOC)—), 2.80 (t, J=7.7 Hz, 2H, —CH₂—NH₂), 1.80-1.50 (m, 4H,—CH₂—), 1.27 and 1.22 (2 s, 18H, t—Bu—).

Cyano Acid 5

The same procedure as for 1 allowed the obtention of the cyano acid 5from 4.

¹H NMR (200 MHz, CDCl₃): d 3.85 (br s, 2H, —CH₂—CO₂H), 3.47 (t, J=6.6Hz, 2H, —CH₂—CH₂—CN), 3.35-3.05 (m, 8H, —CH₂—N(BOC)—), 2.60 (m, 2H,—CH₂—CN), 1.85-1.60 (m, 4H, —CH₂—), 1.46 and 1.44 (2 s, 27H, t—Bu—).

Amino Acid 6

The amino acid 6 (83% yield; purification by chromatography on a silicagel column, eluent methanol/dichloromethane 3/7 then 6/4) was obtainedfrom 5 by the same procedure as compound 2.

¹H NMR (200 MHz, D₂O): d 3.76 and 3.73 (2 s, 2H, —CH₂—CO₂H), 3.25-2.75(m, 12H, —CH₂—N(BOC)— and —CH₂—NH₂), 1.85-1.50 (m, 6H, —CH₂—), 1.28 and1.23 (2 s, 27H, t—Bu—).

Cyano Acid 7

The same procedure as for 1 allowed the obtention of the cyano acid 7from 6.

¹H NMR (200 MHz, CDCl₃): d 3.95 and 3.87 (2 br s, 2H, —CH₂—CO₂H), 3.47(t J=6.5 Hz, 2H, —CH₂—CH₂—CN), 3.40-3.05 (m, 12H, —CH₂—N(BOC)—), 2.61(m, 2H, —CH₂—CN), 1.90-1.60 (m, 6H, —CH₂—), 1.47, 1.45 and 1.44 (3 s,36H, t—Bu—).

Amino Acid 8

The amino acid 8 (71% yield; purification by chromatography on a silicagel column, eluent methanol/dichloromethane 1/9 then 3/7) was botainedfrom 7 by the same procedure as compound 2.

¹H NMR (200 MHz, D₂O): d 3.76 and 3.73 (2 s, 2H, —CH₂—CO₂H), 3.25-2.75(m, 12H, —CH₂—N(BOC)— and —CH₂—NH₂), 1.85-1.50 (m, 6H, —CH₂—), 1.28 and1.23 (2 s, 27H, t—Bu—).

Acid 9

Ditertiobutyldicarbonate (1.19 g, 5.45 mmoles) dissolved in CH₂Cl₂ (5ml) was added to a solution of compound 8 (3.20 g, 4.55 mmoles) andtriethylamine (0.95 ml, 6.83 mmoles) in CH₂Cl₂ (45 ml). The mixture wasstirred for 16 h at room temperature, then acidified to pH 3 with HCl 5%and extracted twice with CH₂Cl₂ (30 ml). The organic phase was washedwith watter (20 ml), dried over sodium sulfate, and concentrated to givea colourless oil which was purified by silica gel column chromatography(methanol/dichloromethane 5/95 then 10/90) to give compound 9 (3.37 g,92%).

¹H NMR (200 MHz, CDCl₃): d 3.85 (m, 2H, —CH₂—CO₂H), 3.45-3.05 (m, 16H,—CH₂—N(BOC)—), 1.85-1.60 (m, 8H, —CH₂—), 1.45, 1.44 and 1.43 (3 s, 36H,t—Bu).

Ester 10

Dicyclohexylcarbodiimide (0.53 g, 2.59 mmoles) in dry dichloromethane (1ml) was added to a solution of the acid 9 (1.60 g, 1.99 mmoles), of(S)-(+)-2,2-dimethyl-1,3-dioxolane-4-methanol (0.34 g, 2.59 mmoles) andof 4-(dimethylamino)pyridine (24 mg, 0.2 mmole) in dry dichloromethane(4 ml). The reaction mixture was stirred for 16 h at room temperature.Then the precipitate of dicyclohexylurea was removed by filtration andthe filtrate was concentrated in vacuo and chromatographed on a silicagel column (eluent ether/hexane 5/5 then 6/4) to give the ester 10 (1.73g; 95%)

¹H NMR (200 MHz, CDCl₃): d 4.35-4.04 (m, 4 H), 3.99-3.92 (2 S, 2H,—CH₂—CO), 3.74 (m, 1H), 3.30-3.00 (m, 16H, —CH₂—N(BOC)—), 1.85-1.55 (m,8H, —CH₂—), 1.46, 1.45, 1.44 and 1.42 (4 s, 48H, t—Bu— and Me—), 1.36(s, 3H, Me—).

Dihydroxyester 11

A solution of ester 10 (1.55 g, 1.69 mmoles) and of 1H hydrochloric acid(0.68 ml) in methanol (29 ml) was stirred for 16 h at room temperature.Triethylamine (1 ml) was then added to the solution until neutral.Evaporation in vacuo and silica gel column chromatography (eluent:methanol/dichloromethane 5/95) gave the dihydroxyester 11 (1.23 g; 83%)as a colourless oil.

¹H NMR (200 MHz, CDCl₃): d 4.25 (m, 2H, —CH₂—OCO—), 4.00-3.40 (m, 5H,CH—OH, —CH₂OH, and —CH₂—CO₂—), 3.40-3.00 (m, 116H, CH₂—N(BOC)—),1.90-1.6 (m, 8H, —CH₂—), 1.46, 1.45, 1.44 and 1.42 (4 s, 45H, t—Bu—).

Triester 12

Dicyclohexylcarbodiimide (0.71 g, 3.42 mmoles) in dry dichloromethane (1ml) was added to a solution of dihydroxyester 11 (1.00 g, 1.14 mmoles),oleic acid (0.97 g, 3.42 mmoles) and 4-(dimethylamino)pyridine (14 mg,0.11 mmole) in dry dichloromethane (3 ml). The reaction mixture wasstirred for 16 h at room temperature. Then the precipitate ofdicyclohexylurea was removed by filtration and the filtrate wasconcentrated in vacuo and chromatographed on a silica gel column(eluent: ether/hexane 4/6) to give the triester 12 (754 mg; 47%) ascolourless oil.

¹H NRM (200 MHz, CDCl₃): d 5.34 (m, 4H, —CH═), 5.26 (m, 1H, CH—OCO—),4.40-4.05 (m, 4H, —CH₂—OCO—), 3.95 and 3.89 (2 m, 2H, —N(BOC)—CH₂—CO₂—),3.35-3.00 (m, 16H, —CH₂—N(BOC)—), 2.31 (t, J 7.5 Hz, 4H, —CH₂—CO₂—),2.01 (m, 8H, allylic H), 1.85-1.50 (m, 12H, —CH₂—), 1.46, 1.44, 1.43 and1.41 (4 s, 45H, t—Bu—), 1.30 and 1.27 (2 br s, 44H, —CHI-), 0.88 (t,J=6.4 Hz, 6H, Me—).

Cationic Glycerolipids pcTG56

The triester 12 (0.52 g, 0.37 mmole) in dry dichloromethane (1 ml) wastreated for 3 h with a 1/1 mixture of trifluoroacetic acid and drydichloromethane (74 ml) at 0° C. Hexane (100 ml) was then added and themixture was evaporated in vacuo to leave a thin film which was suspended(vortex) in distilled ether. Filtration gave a white powder which waswashed with ether and dried in vacuo to give the lipid 13 (510 mg; 93z).

¹H NMR (200 MHz, CDCl₃—CF₃CO₂D): d 5.36 (m, 5H, —CH═ and CH—OCO—),4.60-4.15 (m, 4H, —CH₂—OCO—), 4.00 (s, 2H, —NH₂ ⁺—CH₂—CO₂—), 3.45-3.10(m, 16H, —CH₂—NH₂ ⁺—), 2.41 (t, J=7.5 Hz, 4 H, —CH₂—CO₂—), 2.28 (m, 8H,—CH₂—CH₂—NH₂ ⁺—), 2.01 (m, 8H, allylic H), 1.61 (m, 4H, —CH₂—CH₂—CO₂—),1.30 and 1.27 (2 4, 44 H, —CH₂—), 0.87 (t, J=6.4 Hz, 6H, Me—).

B. Formulation of DNA-Lipid Complexes Containing pcTG56

DNA-lipid complexes were prepared with a final concentration of 0.5 or 1mg/ml DNA at charge ratio 5 (ratio between positive charges carried bythe cationic lipid and negative charges carried by the DNA) with anequimolar amount of dioleyl-phosphatidylethanolamine (DOPE). The amountof cationic lipid to be added was determined based on its molecularweight, the number of positive charges per molecule and the desiredcharge ratio. As an example, to obtain a complex of pcTG56/DOPE at acharge ratio 5 and a final DNA concentration of 0.5 mg/ml, the followingcalculation applies 0.5 mg/ml DNA correspond to a concentration of0.5/330 mmoles/ml=1.5 mmoles/ml negative charges (330Da is taken as theaverage molecular weight of a nucleotide). To obtain a complex at chargeratio 5, the concentration of positive charges must be 7.5 mmol/ml(molecular weight of pcTG56 in the form of its trifluoroacetate salt:1476 g/mol; 5 positive charges per molecule) or 1.5 mmol/ml pcTG56 (2.2mg/ml). To reach an equimolar concentration, 1.5 mmol of DOPE were added(molecular weight 744 g/mol; final concentration of 1.1 mg/ml).Distearoyl-phosphatidylethanolamine (DSPE) coupled to polyethyleneglycolof an average molecular weight of 5000 Da (PEG5000) (Avanti PolarLipids, Alabaster, Ala., U.S.A.; Ref. 880220) (average molecular weighttaken to be 5750 g/mol) was added to reach the required finalconcentrations of 2, 5 or 10 mol % with respect to the total amount oflipid.

Lipids were mixed from their respective solutions in chloroform/methanol(1/1). The lipid solution was dried (200 mbar, 45° C., 45 min) withvortexing (40 revolutions per minute) (Labconco, Rapidvap, Uniequip,Martinsried, Germany) and the lipid film was taken up indimethylsulfoxide (DMSO)/ethanol (1/1). As an example, 2.2 mg pcTG56 and1.1 mg DOPE were taken up in 45 ml dimethylsulfoxide/ethanol. 175 ml 20mM (N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]) (HEPES), pH7.5 were added to reach a final concentration of 10 mg/ml in cationiclipid. 500 ml plasmid DNA (pTG11033; 1 mg/ml in 10 mM(tris)[hydroxymethyl]aminomethane) (Tris), 1 mmethylenediaminetetraacetic acid (EDTA), pH 7.5) were diluted with 280 ml20 mM HEPES, ph 7.5 220 ml of the above lipid suspension were added tothis solution by rapid aspiration-pipetting (10-times) to obtain 1 ml ofthe final complex at a DNA concentration of 0.5 mg/ml and a charge ratioof 5. Preparations at 1 mg/ml DNA with and without DSPE-PEG5000 werereconstituted using the same method by adjusting the amounts of DNA,lipids and stabilizer accordingly. The obtained lipid-DNA complexes werestored at 4° C.

Results

Particle size of the cationic lipid-nucleic acid complexes was measuredby photon-correlation spectroscopy and is given as the mean value weightnormalized (see Example 1). Table 2 summarizes the results of thisexperiment.

TABLE 2 Influence of the DNA concentration and of the % of DSPE-PEG5000on particles size stability Preparation Charge Day after preparationRatio Mean Particle Size (nm) (storage 4° C.) +/− Day 1 Day 7 Day 10 1mg/ml DNA; 2 mol % 5 234 — — DSPE-PEG5000 1 mg/ml DNA; 2 mol % 5 96 — —DSPE-PEG5000 20% DMSO 0.5 mg/ml DNA; 2 mol % 5 150 141 131 DSPE-PEG50000.5 mg/ml DNA; 5 mol % 5 66 60 105 DSPE-PEG5000 0.5 mg/ml DNA; 10 mol %5 68 69 75 DSPE-PEG5000

The results shown above extend our previous findings in that:

a) lipid-DNA complexes can be formed with higher (1 mg/ml) DNAconcentration (previously 0.2 mg/ml);

b) that DSPE-PEG is compatible with lipids of other structural classessuch as cationic glycerolipids (pcTG56), and

c) that DSPE-PEG5000 can replace DSPE-PEG2000.

In addition, they show that our methodology is compatible with otheradditives such as DMSO, which may enhance in vivo gene transfer.

What is claimed is:
 1. A method for preparing an homogenous suspensionof stable lipid-nucleic acid complexes or particles, comprising: a)combining one or more cationic lipids, one or more colipids, and one ormore stabilizing additives to form a lipid suspension, b) combining thelipid suspension with a nucleic acid to form a complex or a particle,and optionally c) subjecting the complex or particle to a sizingprocedure.
 2. The method of claim 1, further comprising the step ofsubjecting the lipid suspension to a sizing procedure to form a lipidsuspension of particles of homogenous size.
 3. The method of claim 1,wherein the sizing procedure comprises extruding the lipid-nucleic acidcomplexes or particles through a membrane of defined pore diameter. 4.The method of claim 3, wherein the lipid-nucleic acid complexes orparticles are extruded through membranes having pore sizes in the rangeof 50 to 500 nm.
 5. The method of claim 1, wherein the lipid-nucleicacid complexes or particles in the homogeneous suspension have aparticle size of 500 nm or less.
 6. The method of claim 1, wherein thelipid-nucleic acid complexes or particles in the homogeneous suspensionhave a particle size of 200 nm or less.
 7. The method of claim 1,wherein the cationic lipid or lipids are selected from the groupconsisting of spermidine-cholesterol, spermine-cholesterol,3β[N-(N′,N′-dimethylaminoethane)-carbamoyl] cholesterol,dioctadecylamidoglycylspermine, and mixtures thereof.
 8. The method ofclaim 1, wherein the colipid is dioleoylphosphatidylethanolamine.
 9. Themethod of claim 1, wherein the stabilizing additive is polyethyleneglycol coupled to a colipid.
 10. The method of claim 1, wherein thestabilizing additive is selected from the group consisting ofperfluorinated or partially fluorinated alkyl chains coupled to colipid.11. The method claim 1, wherein the stabilizing additive ispolyglucuronic acid coupled to a colipid.
 12. The method of claim 1,wherein the stabilizing additive isdistearoylphosphatidylethanolamine-polyethylene glycol.
 13. The methodof claim 9, wherein the moiety is a phospholipid.
 14. The method ofclaim 13, wherein the moiety is a zwitterionnic phospholipid.
 15. Themethod of claim 1, wherein the nucleic acid is selected from the groupconsisting of genomic DNA, cDNA, synthetic DNA, RNA, mRNA, ribozymes,antisense RNA and oligonucleotides.
 16. The method of claim 15, whereinthe nucleic acid comprises a plasmid.
 17. An homogenous suspension ofstable lipid-nucleic acid complexes or particles, produced by: a)combining one or more cationic lipids, one or more colipids, and one ormore stabilizing additives to form a lipid suspension, b) combining thelipid suspension with a nucleic acid to form a complex or a particle,and optionally c) subjecting the complex or particle to a sizingprocedure.
 18. The suspension of claim 17, wherein the lipid suspensionof step a) is subjected to a sizing procedure to form a lip suspensionof particles of homogenous size.
 19. The suspension of claim 17, whereinthe sizing procedure comprises extruding the lipid-nucleic acidcomplexes or particles through a membrane of defined pore diameter. 20.The suspension of claim 17, wherein the cationic lipids include mixturesof cationic lipids.
 21. The suspension of claim 17, wherein thecomplexes or particles in the homnogeneous suspension have a particlesize of 500 nm or less.
 22. The suspension of claim 17, wherein thecomplexes or particles in the homogeneous suspension have a particlesize of 200 nm or less.
 23. A lipid-nucleic acid complex comprising oneor more cationic lipids, one or more colipids, one or more stabilizingadditives, and a nucleic acid component.
 24. The complex of claim 23,wherein the complex has a particle size of 500 nm or less.
 25. Thecomplex of claim 23, wherein the complex has a particle size of 200 nmor less.
 26. A pharmaceutical composition comprising the suspension ofclaim 17 and a pharmaceutically acceptable carrier.
 27. A pharmaceuticalcomposition comprising the complex of claim 23 and a pharmaceuticallyacceptable carrier.
 28. A method for the delivery of a nucleic acid tothe cells of an individual in need of such nucleic acid, comprisingadministering the composition of claim 26 to the cells of such anindividual.
 29. The method of claim 28, wherein the nucleic acid isselected from the group consisting of genomic DNA, cDNA, synthetic DNA,RNA, mRNA, ribozymes, antisense RNA and oligonucleotides.
 30. The methodof claim 29, wherein the nucleic acid comprises a plasmid.
 31. Themethod of claim 2, wherein the sizing procedure comprises extruding thelipid suspension through a membrane of defined pore diameter.
 32. Themethod of claim 31, wherein the lipid suspension is extruded throughmembranes having pore sizes in the range of 50 to 500 nm.
 33. Thesuspension of claim 18, wherein the sizing procedure comprises extrudingthe lipid suspension through a membrane of defined pore diameter. 34.The suspension of claim 19, wherein the lipid-nucleic acids complexes orparticles are extruded through membranes having pore sizes in the rangeof 50 to 500 nm.
 35. The suspension of claim 33, wherein the lipidsuspension is extruded through membranes having pore sizes in the rangeof 50 to 500 nm.